Bcl-2 family proteins use complex intra-family interactions to regulate the intrinsic apoptotic pathway. In particular, anti-apoptotic Bcl-2 famil members (A1, Bcl-2, Bcl-xL, Bcl-w and Mcl-1) inhibit apoptosis by sequestering pro-apoptotic Bcl-2 family members (Bak, Bax, Bid, Bim and Puma). Thus, the balance of pro- to anti-apoptotic Bcl-2 proteins regulates the cellular life-death switch. In cancers, sustained overexpression of anti-apoptotic Bcl-2 family members promotes tumor cell survival. Further, anti-apoptotic Bcl-2 proteins often drive resistance of cancers to chemotherapies and targeted therapies. BH3-mimetics, small molecules designed to sequester the activity of anti-apoptotic Bcl-2 family members, were designed to combat therapeutic resistance caused by anti-apoptotic Bcl-2 proteins. In breast cancers, BH3-mimetics increase treatment-induced tumor cell killing in some but not all tumors. The mechanisms underlying resistance to these BH3-mimetics remain unclear. Our preliminary data suggest that human-derived breast cancer cell lines with MCL1 amplification display limited sensitivity to ABT-263, a BH3-mimetic targeting Bcl-2, Bcl-xL and Bcl-w, and up regulate Mcl-1 protein expression upon treatment with ABT-263. Mcl-1 knock-down increased sensitivity of breast cancer cells to ABT-263, while ectopic Mcl-1 expression enhanced ABT-263 resistance. Given the clinical impact of the Bcl-2 family in breast and other cancers, it is critical to investigate further the role of Mcl-1 in ABT-263 resistance, which we wil study in Aim 1. We further propose to study how Mcl-1 affects response of breast cancers to estrogen deprivation. Approximately 65% of breast cancers are dependent upon estrogen receptor-a (ERa) signaling, and thus are treated with ERa-targeting agents. The impact of Mcl-1 remains under-studied in this context. Our preliminary data show that Mcl-1 levels increase upon long term estrogen deprivation (LTED), a model used to mimic the estrogen-depleted conditions caused by treatment with aromatase inhibitors. We designed to combat therapeutic resistance caused by anti-apoptotic Bcl-2 proteins in Aim 2. Together, these studies will determine if Mcl-1 inhibitors, including a novel Mcl-1- specific BH3-mimetic developed by our collaborator, could be exploited clinically to improve tumor cell killing and to increase patient survival.